One of the most important problems in healthcare today is the early detection of disease states, whether one is trying to detect the onset of cancer or a specific viral infection. Most current assay technologies involve microliters or milliliters of sample and have moderate detection limits that require a significant analyte concentration. On the other hand, a single drop of blood or saliva may contain a complete chemical signature of particular states of wellness or disease-based on proteins or other molecules that are expressed during the state of distress are expressed in trace amounts. To detect trace analyte levels with such small sample volumes, a detection limit in the single-molecule range is required.
With the latest advances in mass spectrometry that provide high ionization efficiency and efficient ion collection optics, the state of the art is one step closer to achieving robust assays with low detection limits. However, in order to deliver quantitative assays with low detection limits in the single-molecule regime, two problems need to be solved.
The first problem associated with current mass spectrometry methods is that the process of ionization is not completely reproducible. Ionization is a necessary step that occurs after volatilization of the molecules of interest, but prior to the detection process based on the mass to charge ratio. Due to nonlinear fluctuations in the ionization yield from sample to sample, noise is introduced in the detection process that results in an elevated limit of detection. As a result, current methods, despite their high sensitivity, are not considered sufficiently quantitative for clinical diagnostics.
The two most common methods of introducing molecules to the gas phase are matrix-assisted laser desorption (MALDI) and electrospray. The very act of ionization is highly nonlinear for both methods of ionization. In the case of MALDI, there are complex chemical reactions involving proton exchange that are inherently nonlinear and occur under nonequilibrium conditions. Similarly, electrospray involves the charging of droplets that leads to statistical fluctuations that are amplified by the ensuing coulomb explosion process that strips off solvent molecules in the electrospray method. The number of ions generated for a given amount of material introduced to the gas phase by either method is irreproducible due to the highly nonlinear nature of the ion generation step. Small differences in conditions translate to large variations in the detected species.
To achieve a quantitative clinical diagnostic assay with a low coefficient of variation and a low limit of detection, it is essential for any measurement to be highly reproducible. To date, the nonlinear ionization process has prevented mass spectrometry from achieving both single molecule detection limits and the required level of quantification in order to catch disease signatures at the earliest possible stage of development with minute quantities of tissue or bodily fluids.
A second problem is that the very act of generating a gas phase sample of the specimen of interest, as required for mass spectrometry may lead to extensive fragmentation of the parent molecule. The various proteins in the body are all very similar, being composed of the same nucleotides and amino acid monomers. Excessive fragmentation makes it impossible to make unique assignments to molecular species. As long as one has a parent ion component, as a reference point, fragmentation patterns can aid in identification of the parent molecule.
Unfortunately, the fragmentation pattern needs to be highly reproducible and unique to a given molecule. If there is more than one possible molecular species present, then fragmentation leads to overlapping mass peaks and loss of identification. In order to identify proteins within an unpurified sample or directly from tissue, the fragmentation of the parent molecule be minimized as much as possible to avoid congestion in the fragmentation patterns and the fragmentation needs to induced in a controlled and known way. Otherwise, there will be too much overlap of common fragments to decipher the fragmentation pattern and identify the molecule of interest (e.g. a protein). Current mass spectrometry methods are unable to achieve such control over fragmentation, and therefore require the aforementioned onerous pre-analytic separation steps. For this reason, mass spectrometry has principally been confined to identification of highly purified samples.
This problem is exacerbated by the natural propensity for certain molecules to form aggregates, and is particularly severe in proteomics research where one is interested in quantitative information on the expression of certain proteins. However, these proteins often occur naturally in complexes or a distribution of isolated molecules and multiple complexes with different proteins involving complicated equilibria with several other protein complexes. Within a mass spectrometer, the ionization of protein complexes leads to fragmentation patterns or spurious mass to charge ratios that mask the identity of the molecule(s) of interest.
For this reason, most of the time involved in performing a proteomic assay involves the pre-analytic purification of proteins, which is achieved by batch processing relatively large amounts of material and using various complex and time consuming steps (such as chromatography and electrophoresis) to separate the initial mixture into highly purified protein components. The purified proteins are then injected into a mass spectrometer for identification. The purification step is required to enable identification of the protein by ensuring all mass fragments are from the same molecule.
Even with this step, quantification of the amount of protein present in the initial sample is simply not possible, for the reasons discussed above. Further, the need to purify proteins before subjecting the sample of interest to detection greatly reduces the efficiency or throughput of mass spectrometry. As a result, despite the inherently high sensitivity of mass spectrometry, large sample volumes are required in order to compensate for the downstream losses in the purification steps. To achieve the ultimate detection limits of mass spectrometry, in situ isolation and separation of molecular species of interest is required.
What is therefore needed is an improved method of sample preparation for mass spectrometry that delivers reproducible ionization and minimal molecular fragmentation required for quantitative and sensitive assays.